1. Field of the Invention
The invention relates to a device for treating a porous filtration medium having a receiving unit consisting of a receiving part and a base part, wherein the porous filtration medium can be received and lifted by the receiving part from a lower part of a filtration device, and the receiving part with the porous filtration medium can be mounted on the base part, and wherein the receiving part is formed so as to be latchable to the base part.
2. Description of the Related Art
The invention further relates to a method for treating a porous filtration medium with a receiving unit of a device, said receiving unit consisting of a receiving part and a base part                in which the receiving part is placed on the filtration medium that is arranged in a lower part of a filtration device and exposed to a liquid sample, wherein a fixing edge arranged in the receiving part is connected to an edge of the filtration medium,        in which the receiving part with the connected filtration medium is lifted from the lower part and placed on the base part, whereby the receiving part and the base part are latched together.        
Primarily microbiological methods, which detect individual microorganisms by using cultivation steps, are currently used for routine investigations. These methods are, however, very time-consuming and may take several days to detect contamination of the aqueous medium. Modern, rapid methods for detecting microorganisms, such as real-time PCR, antibody assays or analytical microarrays, facilitate the quick detection of microbial contamination. But first, in order to lower the detection limits in these detection methods and ideally also to be able to detect a single microbe, fast, effective enrichment steps are required in order to concentrate a large sample volume of up to several liters into a few hundred microliters. The concentrated sample allows for better handling with less consumption of reagents and can be processed according to any of the subsequent detection methods.
Various treatment methods using porous media such as filters and membranes have become established in the analysis of liquids and gases. For instance, a filtration method for enriching and concentrating dissolved or particulate substances has been established. Such concentration is generally necessary if the concentrations of the contaminants are too low to perform direct evaluations. Filtration methods are the precursor of further analytical methods, such as visual evaluations, as well as of further physical and chemical reactions for signal amplification.
Only small sample volumes can be used for newer, more sensitive analytical methods, e.g. polymerase chain reaction (PCR), and the preparation of the samples used in such methods. Filtration membranes with diameters of 47 mm or 25 mm are typically used to filter for concentration purposes the sample volumes of more than 100 ml that are typical in many cases. Even after filtration, when the substances or particles are present in concentrated form on the filtration membrane, the membrane-bound particles cannot be conveyed directly to analysis because of the size of the membrane. It is necessary to transfer the retained substances to a sample volume, which ideally should not exceed 1 ml, in order to create a sample preparation for the subsequent analysis in standard reaction vessels that will fit into tabletop centrifuges, which are typically part of the standard equipment available in any laboratory.
A device and a method for treating a porous filtration medium with a receiving unit consisting of a receiving part and a base part are known from WO 2011/057707 A2. With the receiving part the porous filtration medium can be lifted off from a lower part of a filtration device, and the receiving part with the porous filtration medium can be placed on the base part, the receiving part and the base part being designed so as to be reversibly connected to each other. The known device, which has basically proved itself in practice, serves to transfer filtered substances by means of back-flushing from a filter (filtration medium) to a receiving vessel connected to the receiving part.
A disadvantage of this device is that, because of the distribution of pore sizes in most membrane filters, numerous particles are separated out not on the surface of the membrane filter but rather in deeper layers so that a quantitatively complete back-flushing of the particles is not possible. Non-specific adsorption events of the retained particles on the membrane also intensify this problem.
A culture-medium unit and a method for receiving a filter from a filtration device are known from DE 10 2008 005 968 A1. This culture-medium unit consists of a cover or receiving part, which forms the actual transfer unit, and a lower part filled with a culture medium. The upper part, which is designed to serve as a receiving part, has a fixing edge which can be connected to an edge of the filter by means of an adhesive bond in order to remove the filtration medium from the filtration device or treatment device.
Also known from DE 10 2008 005 968 A1 is a method for the microbiological examination of liquid samples, in which a cover or receiving part of a culture-medium unit is placed on a filter, designed as a membrane filter and having a fixing edge, which is arranged in a lower part of a filter device or treatment device. In this case the fixing edge of the receiving part is connected to an edge of the filter by means of an adhesive layer. The receiving part with the filter is then lifted from a filter support in the lower part of the filter device and placed on the surface of a nutrient medium arranged in the lower part of a culture-medium unit, and the cover or the receiving part covers the dish-shaped lower part.
However, a disadvantage of the known filtration units and the corresponding methods, which have proved their usefulness for classic, microbiological membrane applications in which only particles are removed or in which established colonies are visually evaluated in the field of microbiology, is the fact that after filtration the retained particles or their constituent substances can no longer be removed from the membrane by flushing in such a manner that highly concentrated suspensions result.
The dissolving of a porous filtration medium with the goal of performing a PCR analysis of the constituent substances is known from JP 2012-019723 A, from L. J. DiMichele (Am. Soc. Brew. Chem., 1993, Vol. 51, No. 2, pp. 63-66), from K. Nakamura (Journal of Aerosol Research, 2003, Vol. 18, No. 3, pp. 177-180), and from K. Stark (Applied and Environmental Microbiology, 1998, Vol. 64, No. 2, pp. 543-548).
However, a disadvantage of the method described in each of these documents is the high risk of contamination since the filtration medium must be picked up, folded and transferred to a reaction vessel (generally a 1.5- to 2-ml vessel) using tweezers. The subsequent addition of a solvent for the membrane also represents a risk of contamination in an open system.
Also known from JP 2012-019723 A is the method of using acetone to dissolve cellulose membranes on which microorganisms are fixed and adding aqueous buffering solutions in order to derive a solution containing the microorganisms.
A disadvantage of this method is that this process again precipitates out some cellulose in fiber form and that the cellulose fibers have the undesired effect of binding to portions of the microorganisms or the DNA, thus making the quantitative analysis of the microorganisms more difficult. Certain additives, e.g. cetyltrimethylammonium bromide, are added to reduce the undesirable adsorption of the microorganisms or DNA on the fibers. But a complete quantitative analysis cannot be achieved.
Further, a method is known from L. J. DiMichele (Am. Soc. Brew. Chem., 1993, Vol. 51, No. 2, pp. 63-66) for dissolving polycarbonate membranes, on which microorganisms are fixed, in a mixture of water and chloroform (200 μl water and 300 μl chloroform) with the goal of enriching the microorganisms in the aqueous phase. After the aqueous phase is transferred to a new vessel, the microorganisms are pelletized by means of centrifugation. A washing step follows and then the PCR.
A disadvantage of this method is that microorganisms do not in practice accumulate in the upper aqueous phase. Rather, the microorganisms tend to sediment in the organic phase (lower phase) or in the boundary layer so that the complete recovery of the microorganisms cannot be achieved with this method. Furthermore, the described method does not have a lysis step to disrupt the microorganisms so that it must be assumed that numerous intact cells are used for the PCR and thus amplification will not be possible for a large portion of the DNA.
Further, a method is known from K. Stark (Applied and Environmental Microbiology, 1998, Vol. 64, No. 2, pp. 543-548) for dissolving in chloroform polyethersulfone membranes on which microorganisms are fixed. TE buffers are then added and there follows a ten-minute extraction of DNA into the aqueous phase under agitation at room temperature. The aqueous solution is then subjected to DNA precipitation with alcohol before the PCR evaluation is performed.
A disadvantage of this method is that only a small proportion of the DNA can be extracted into the aqueous phase because there is no prior lysis step to disrupt the microorganisms and thereby make the DNA freely accessible. Rather, using this method results in the sedimentation of the still intact microorganisms into the lower organic phase or into the boundary layer between the organic and aqueous phases.
A method is also known from K. Sen (Applied and Environmental Microbiology, 2007, Vol. 73, No. 22, pp. 7380-7387) for folding the filtration medium with a pair of tweezers and transferring it into a reaction vessel. The membrane does not undergo a dissolving step but rather is only rinsed with, for instance, a commercial lysis buffer, or the membrane is mechanically stressed by vortexing it together with grinding balls, which serves to disrupt the cells. K. Sen uses various commercial kits for DNA isolation.
A disadvantage of the methods described is not only the increased risk of contamination from folding and transferring the membrane with tweezers, but also that it is not possible to completely rinse the microorganisms from the membrane because microorganisms are frequently also separated out in deeper layers of the membrane, and also that non-specific adsorption may occur on the membrane so that a superficial flushing step is not effective. Another complicating factor is that with a membrane that has been folded up small in a reaction vessel, no targeted back-flushing of the membrane is possible, instead only undirected mixing or vortexing of the membrane and flushing solution can be accomplished.
From WO 2012/031156 A1 a filtration device is known which enables work to be carried out in a contamination-free setting by retaining the filtration medium (diameter of filtration surface: 13 mm) in the sealable device and disrupting the cells directly on the membrane using grinding balls and vortexing. The free DNA passes through the membrane in a subsequent filtration step.
A disadvantage of this method is that the quantitatively complete disruption of the cells is not possible because a large portion of the microorganisms generally penetrates into the deeper layers of the membrane and is thereby shielded from the grinding balls. This buffering effect also has a negative influence on the degree of disruption of the microorganisms because a large proportion of the impacts are absorbed by the membrane. In addition, the subsequent step of filtering the DNA through the membrane will be incomplete because DNA has a tendency to form non-specific bonds, in this case on the membrane. Moreover, the diameter of the filtration surface is limited to 13 mm in this device as a result of its compatibility with common centrifuge models and adapters. However, this small diameter of the filtration medium results in considerably longer filtration times for large sample volumes.
EP 2 402 456 A1 discloses a method for analyzing microorganisms in water samples in which a water sample containing microorganisms is injected by a first syringe into a Minisart® syringe filter with a cellulose-ester membrane to retain the microorganisms. After the syringe is removed, one end of the syringe filter is connected to a receiving vessel, while the other end of the syringe filter is connected to a second syringe filled with a polar aprotic solvent such as DMSO (dimethyl sulfoxide). DMSO is injected into the syringe filter until the pressure point is reached in order to dissolve the membrane with the retained microorganisms and collect the solution in the receiving vessel. Centrifugation of the solution in the receiving vessel is followed by cell lysis and further microbiological analytical steps, such as PCR.
Disadvantages of the method known from EP 2 402 465 A1 are the successive handling with two different syringes—one of which contains a water sample and the other of which contains the solvent for the cellulose-ester membrane—and the fact that when injecting the DMSO into the syringe filter the injection pressure must not be too high, i.e. it must be less than the pressure point, so that the syringe filter is not damaged.
The task of the present invention is, therefore, to provide a device and a method with which it is possible to transfer a porous filtration medium including retained microorganisms easily, safely and without risk of contamination to a receiving vessel in order to make the sample quantitatively completely accessible for DNA extraction and molecular biological analysis.